The ability to modify microorganisms and cells of higher organisms by genetic engineering has made it possible to change certain of their specific characteristics and thereby alter the response of those organisms to various agents. Of particular interest are the responses of organisms to agents used because of their cytotoxic effect. For example, many compounds used in agriculture are directed to the killing of pests, weeds, or the like. Often these compounds can have a relatively long residence time or extended residue in the plants subjected to treatment by the compound.
In many situations it is desirable to differentiate the species to be retained from the species to be killed. For example, it is often necessary to selectively destroy weeds, yet have minimal impact on the economically valuable crop plants. For the most part, broad-spectrum herbicides have a sufficiently adverse effect on crops that their use must be limited to emergent use or careful postemergent application.
Some weed species are simply resistant to today's herbicides, increasing the importance of developing the production of effective herbicides. Moreover, as some weed species are controlled, competition is reduced for the remaining tenacious weed species. The development of genetically engineered herbicide-resistant crop plants could significantly improve weed-control by allowing fields to be treated with a single, concentrated application of the herbicide. Therefore, a one-step procedure could eliminate costly and perhaps ineffective repeated low-dosage herbicidal treatments, such as have been required in the past to avoid damaging conventional crops, but which may have also induced the emergence of spontaneous herbicide-resistant weeds. Herbicides with greater potency, broader weed spectrum and more rapid degradation after application would avoid the problematic persistence of the chemical herbicide in the soil, such as typically results from frequently repeated applications, and which prevents rotation of crops sensitive to that herbicide.
Certain herbicides, while not used directly to control weeds in field crops, are used as total vegetation control agents to eliminate weeds entirely in certain right-of-way or industrial situations. However, these herbicides may be deposited by natural means, such as water run-off, onto areas where economically important crops are growing. As a result sensitive field crops may be killed or their growth seriously inhibited. It is therefore highly desirable to be able to modify viable cells to make them resistant to stressful cytotoxic agents.
Isoxaben (-3[1-ethyl-1-methylpropyl]-5-isoxazolyl-2,6,dimethoxybenzamide), 2,6-dichlorobenzonitrile (DCB) and thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidnone (TZ), are structurally diverse herbicides. On the basis of biochemical studies of mode of action, their primary target site has been proposed to be the enzyme cellulose synthase, which catalyses the synthesis of cellulose, a major component of plant cell walls. However, the precise target for these herbicides has not been previously described.
Isoxaben (EL-107, Flexidor, Gallery) is a preemergence, broad leaf herbicide used primarily on small grains, turf and ornamentals. The compound is extremely active with an I50 for Brassica napus of 20 nM (Lefebvre et al., 1987). Isoxaben inhibits the incorporation of glucose into the cellulose-rich, acid-insoluble fraction of isolated walls and is an extremely powerful and specific inhibitor of cell wall biosynthesis (Heim et al., 1990b; Corio-Costet et al., 1991 b). Cell wall-fractionation studies have revealed that the herbicidal action of isoxaben can be explained entirely by its effect on cellulose biosynthesis (Heim et al., 1991). Its probable mode of action is to directly inhibit cellulose synthesis, because resistant cell lines show an unaltered uptake or detoxification of the herbicide (Heim et al., 1991) and only two genetic loci in Arabidopsis thaliana, termed ixrA (=ixr1) and ixrB (=ixr2), have been shown to confer resistance (Heim et al., 1989, 1990a). Exhaustive studies have revealed that other cellular processes are unaffected by isoxaben (e.g. seed germination, mitosis, respiration, photosynthesis, and lipid and RNA synthesis, Lefebvre et al., 1987; Corio-Costet et al., 1991a). Treated cells fail to elongate with high fidelity and consequently grow isodiametrically (Lefebvre et al., 1987). This herbicide acts at much lower concentrations (<40×) than dichlobenil, another cellulose synthesis inhibitor (Heim et al., 1990b). Therefore, the properties of isoxaben make it an ideal agent for perturbing the mechanical properties of the primary cell wall.
Thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidnone (TZ) are a new class of N-phenyl-lactam-carbamate herbicides (Sharples et al, 1998). TZ shows potential for selective preemergence control of a range of weed species in soybean and other crops. Susceptible weeds include grasses such as Digitaria spp., Setaria spp., Sorghum spp., and small seeded broad leafed weeds which include Amaranthus spp., and Chenopodium spp. TZ has a similar syndrome of effects on plants as isoxaben. A common mode of action with isoxaben is indicated by the fact that the ixrA1 (=ixr1-1) mutant of Arabidopsis exhibits resistance to both isoxaben and TZ (Sharples et al., 1998).
The herbicide 2,6-dichlorobenzonitrile (dichlobenil, DCB) is an effective and specific inhibitor of cellulose synthesis in algae and plants (Delmer et al., 1987). It has been reported to bind to an 18 kd polypeptide in cotton fiber extracts but no mechanism for its action has been demonstrated and no function for the 18 kd protein has been reported.
A crop made more resistant to isoxaben and thiazolidinone herbicides offers a selective means to control and kill weeds without adversely affecting the crop plant. Clearly then, an understanding of the method by which weeds become resistant to herbicides at the molecular level is essential to establishing a basis for the development of sound weed control programs. The molecular basis underlying the expression of isoxaben and thiazolidinone-resistance had remained undetermined until the present invention. Therefore, identification of the mutation site(s) in the CS gene, which code for the mutant plant's isoxaben and thiazolidinone resistance is of agricultural significance. The isolation of a mutant CS gene, which confers resistance to isoxaben and thiazolidinone in higher plants, would provide an opportunity to introduce isoxaben and thiazolidinone resistance into crop plants by genetic engineering. Isoxaben and thiazolidinones, because of their broad-spectrum activity and low mammalian toxicity, are particularly suited as a type of herbicide to which genetically engineered resistance would be economically important in crop plants. The development of isoxaben and thiazolidinone-resistant crops would provide a reliable and cost-effective alternative to conventional weed management programs.
By modifying crop plant cells by the introduction of a functional gene expressing the isoxaben and thiazolidinone-resistant CS enzyme, one can use isoxaben and thiazolidinones or an analogous herbicide with a wide variety of crops at a concentration which ensures the substantially complete or complete removal of weeds, while leaving the crop relatively unaffected. In this manner, substantial economies can be achieved in that fertilizers and water may be more efficiently utilized, and the detrimental effects resulting from the presence of weeds avoided.
The genetic code is degenerate, meaning that more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any set of similar DNA oligonucleotides. With respect to nucleotides, therefore the term “derivative(s)” is intended to encompass those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.